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Very Late DNA Replication in the Human Cell Cycle Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11246–11250, September 1998 Cell Biology Very late DNA replication in the human cell cycle i R. J. WIDROW*†,R.SCOTT HANSEN‡,HIROSHI KAWAME‡,STANLEY M. GARTLER‡§, AND CHARLES D. LAIRD†§¶ *Molecular and Cellular Biology Program of the University of Washington and Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Seattle, WA 98104; Departments of ‡Medicine, §Genetics, and ¶Zoology, University of Washington, Seattle, WA 98195; and iMolecular Medicine Division, C3-168, Fred Hutchinson Cancer Research Center, Seattle, WA 98104 Contributed by Stanley M. Gartler, July 23, 1998 ABSTRACT G2 was defined originally as the temporal cell cycle (9). We address two questions. Does this very late gap between the termination of DNA replication and the replication occur during what is usually termed G2? What beginning of mitosis. In human cells, the G2 period was proportion of the genome replicates in this late compartment? estimated to be 3–4 h. However, the absence of replicative DNA synthesis during this period designated G2 has never been shown conclusively. In this report, we show that, at MATERIALS AND METHODS some autosomal and X linked loci, programmed DNA rep- Cell Culture. Lymphoblastoid cell lines were grown as lication continues within 90 min of mitosis. Furthermore, described (9). Cell lines used in this study included: FF, derived the major accumulation of cyclin B1, a cell-cycle marker that from a normal male (5); VK and SK, derived from normal is usually ascribed to G2, overlaps extensively with very late females; and 5106, derived from a phenotypically normal male DNA replication. We conclude that the G2 period is much (kindly provided by B. A. Oostra, Erasmus University, Rot- shorter than previously thought and may, in some cells, be terdam, Netherlands, and described below). Fresh blood lym- nonexistent. phocytes were taken from two normal females, isolated by centrifugation over a Ficoll gradient, and cultured at 5 3 106 The eukaryotic cell cycle consists of an orderly series of events cells per ml in RPMI medium 1640 plus 10% fetal bovine in which cells grow, replicate their DNA, and segregate the serum. Cells were stimulated to divide with the addition of 20 duplicated genome to the daughter cells. Our current textbook mlyml phytohemagglutanin for 3 days before BrdUrd labeling. view of the cell cycle was first put forth by Howard and Pelc General PCR Conditions. All PCRs contained 18.2 mlof (1) as encompassing four phases: S phase, the time in which PCR Supermix (GIBCOyBRL), each primer at 2 mM, and 1 ml replicative DNA synthesis occurs; M phase, the time in which of the BrdUrd-labeled DNA isolated from each fraction. mitotic chromosome segregation and cell division proceed; Parameters for all PCRs included denaturation at 95°C for 5 and two gap phases, G1 and G2, that temporally separate S min and 23 cycles that included denaturation at 95°C for 1 min, phase from mitosis. annealing at a temperature described below for each primer The G2 phase was inferred from the length of time between pair for 2 min, and extension at 72°C for 3 min. A final the addition of radiolabeled DNA precursors and the appear- extension was carried out at 72°C for 7 min. PCR products were ance of labeled, condensed mitotic chromosomes (1). Auto- run on 1.2% Tris-base, acetic acid, EDTA (TAE) agarose gels. radiography, however, may not be sufficiently sensitive to Transfer of DNA to Hybond N1 membrane filters was carried detect low levels of replicative synthesis in the G2 phase. The out with 0.4 M NaOH, followed by neutralization with 0.4 M absence of detectable incorporation of radiolabel could thus Na2HPO4. Hybridization was carried out at 65°C in FBI be taken to indicate erroneously the absence of DNA repli- solution (10% polyethylene glycol 8000y7% SDSy1.53 stan- cation. Furthermore, DNA repair synthesis can occur through- dard saline phosphateyEDTAy100 mgyml sheared salmon out the cell cycle (2, 3); replicative and repair DNA synthesis sperm DNA) for at least 3 h. Thereafter, filters were washed are not easily distinguishable by the autoradiographic method. twice in 33 standard saline citrate with 1% SDS, then washed An additional problem is that no cellular marker signaling the twice in 13 standard saline citrate with 1% SDS, and exposed termination of replicative DNA synthesis has been identified. to x-ray film. These features contribute to an inherent lack of both sensitivity The following annealing temperatures were used: G6PD at and specificity for accurately defining the terminal border of 50°C, F9 at 60°C, PGK1 at 62°C, FMR1 at 60°C, inter-Alu 6–84 S phase. at 45°C, inter-Alu 6–82 at 45°C, inter-Alu H-1 at 40°C, and Our studies on fragile sites and the replication timing of inter-Alu H-8 at 40°C. The oligonucleotide primer pairs used FMR1, the gene responsible for the fragile X syndrome, first for PCR of G6PD, F9, and PGK1 have been described (5). The brought our attention to the possibility of replication very close primer pair and corresponding size used for FMR1 are as to mitosis (4). In cell lines derived from patients with fragile follows: fmr69 (59-gagtcaagtggggaagactaagttgc-39) and fmr357 X syndrome, the replication of a large region surrounding the (59-gaactttgtgcataccctctgcttcc-39) (291 bp). The oligonucleo- y FMR1 gene is abnormally late, occurring within the G2 M tide primer pairs employed for allele-specific inter-Alu PCR compartment defined by DNA content (5, 6). Surprisingly, and the corresponding sizes are as follows: inter-Alu 6–84, equally late replication was observed for several loci on the 6–84F (59-agtatccacaggtatctga-39) and 6–84R (59-ttagaaagta- inactive X chromosome in normal female fibroblasts (7, 8), as cattaggcac-39) (240 bp); inter-Alu 6–82, 6–82A (59- well as on the inactive X chromosome in human–hamster cgatatgaagaaaataaaag-39) and 6–82B (59-atctttaatctcatctccat- hybrids (8). In this report, we have determined more precisely 39) (246 bp); inter-Alu H-1, H-1F (59atcccatacatagacca-39) and the timing of late DNA replication in the human cell cycle, by H-1R (59-agtaaagcattgatcca-39) (262 bp); inter-Alu H-8, H-8F using a new, sensitive method for fractionating cells late in the (59-tagcatttgattttcta-39) and H-8R (59-ttctctattattacgga-39) (225 bp). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in †To whom reprint requests should be addressed at: Fred Hutchinson accordance with 18 U.S.C. §1734 solely to indicate this fact. Cancer Research Center, 1100 Fairview Avenue North C3-168, P.O. © 1998 by The National Academy of Sciences 0027-8424y98y9511246-5$2.00y0 Box 19024, Seattle, WA 98109-1024. e-mail: [email protected]. PNAS is available online at www.pnas.org. edu and [email protected]. 11246 Cell Biology: Widrow et al. Proc. Natl. Acad. Sci. USA 95 (1998) 11247 Replication Timing with Cyclin B1 Flow Cytometry. Flow electrophoretically on a 1.3% TAE agarose gel and transferred cytometry was performed as described (9), with a few excep- to a Hybond N1 membrane. Incorporation of radiolabeled tions. DNA content was measured by resuspending the cells in nucleotides into the PCR products amplified with BrdUrd 1 ml of the staining solution as described (5). In some DNA from each replication-timing fraction was quantified by experiments, 20 ml of antibody (FITC-conjugated Cyclin B1 phosphorimager analysis. The percentage of inter-Alu repli- Antibody Reagent Set; PharMingen) was used instead of 0.5 cation ongoing in each cell-cycle fraction was calculated as mg of the antibody with no discernible differences in results. described above, yielding percentages of inter-Alu replication Previously, we have described a method for studying repli- of 1.3, 29, 33, 18, 9.8, 4.8, 2.9, 0.8 for G1 through high cyclin B1, cation timing that uses the incorporation of BrdUrd (5). We respectively. used this method with the following changes: the immunopre- The cell line 5106 (used to obtain the data shown in Fig. 3B) cipitation of BrdUrd-labeled DNA was carried out with 1 mg was derived from a normal male with a large expansion of the of anti-BrdUrd monoclonal antibody and 16.5 mg of rabbit IgG CGG repeat in the FMR1 gene but without associated meth- directed against mouse IgG; lyophilized BrdUrd DNA isolated ylation (kindly provided by B. A. Oostra). We did not detect from each sorting fraction was dissolved in a volume of any differences in the replication-timing profiles of several Tris-EDTA to obtain the equivalent of 400 cells per ml; and specific inter-Alu sequences between this and other cell lines, BrdUrd-labeled CHO-YH21 DNA was not added. All cell confirming that the expansion of the CGG repeat does not fractionation isolates were controlled internally by assaying have a general effect on replication timing (5). In addition, characteristic early and late replicating sequences (for exam- comparable results were obtained by using the normal male ple, see H-8, Fig. 3C). Individual sequence profiles were cell line, FF. obtained in at least two different cell lines. Quantitation of the hybridization signals was performed by densitometry analysis. To calculate the percent of DNA rep- RESULTS lication in the mid- and high cyclin B1 fractions for loci on the Cell-Cycle Fractionation for Replication Timing. To inves- inactive X chromosome, we assumed that the total signal tigate very late DNA replication, we have developed a method constituted the later replication signal.
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